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making P32 probes - making P32 labeled DNA probes for southerns (Jul/08/2005 )

I'm confused about how to make P32 labeled DNA probes for southern. I have the primers and I want to use eukaryotic genomic DNA for a template.
Is it possible to simply run a PCR and include some p32 labeled dNTPs into it?
I noticed that most ppl recommend the random primed PCR labeling method. Is this method useful for my purpose considering that I already have primers that work?


The easiest method is to label one of your primers with kinase and gamma 32P-ATP, 10 µCi/µL, 3.33 µM and 3000 Ci/mmol. Using a 1:1 ratio of label:primer, approximately 50% of the primer (and subsequently, your PCR product) will be labeled. Clean up the reactions with a Sephadex G-25 or G-50 cartridge.

If you use (say 5 uL) of alpha 32P-dATP, 10 µCi/µL, 2.0 µM and 5000 Ci/mmol mixed with the cold 200 uM dATP in a 100 uL reaction, you are mixing 10 pmoles of hot dATP with 20,000 pmoles of cold dATP. Only 1 out of 2000 As in the sequence will be labeled. If your PCR product is 100 bp, then assuming that there are 25 As in the product (there are actually fewer because the primers will not include hot As), then only 1 out of 80 products will be labeled.

Lightly labeled DNA is more stable than heavily-labeled material. It all depends on your desired exposure time and how quickly you will use the labeled probes.